Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
PAF1

Cell type

Cell type Class
Bone
Cell type
U2OS
Tissue
bone
Lineage
mesoderm
Description
osteosarcoma from the tibia of a 15 year old, J. Ponten and E. Saksela derived this line (originally 2T) in 1964 from a moderately differentiated sarcoma, viruses were not detected during co-cultivation with WI-38 cells or in CF tests against SV40, RSV or adenoviruses, mycoplasma contamination was detected and eliminated in 1972, (PMID: 6081590)

Attributes by original data submitter

Sample

source_name
U2OS CSB-KO
cell type
U2OS
genotype/variation
CSB-KO
uv-c dose
no UV
time after uv-c
not applicable
chip-antibody
PAF1; Bethyl (A300-172A)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP-seq: Cells were plated and grown to ~90% confluency and crosslinked with 0.5 mg/mL disuccinimidyl glutarate (DSG; Thermo Fisher) in PBS for 45 min at room temperature. Cells were washed with PBS and crosslinked with 1 % PFA for 20 min at room temperature. Fixation was stopped by adding 1.25 M Glycin in PBS to a final concentration of 0.1 M for 3 minutes at room temperature. Cells were washed with cold PBS and lysed and collected in a buffer containing 0.25% Triton X-100, 10 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0) and 20 mM Hepes (pH 7.6). Chromatin was pelleted in 5 min at 400 g and incubated in a buffer containing 150 mM NaCl, 1 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0) and 50 mM Hepes (pH 7.6) for 10 minutes at 4°C. Chromatin was again pelleted for 5 min at 400 g and resuspended in ChIP-buffer (0.15 % SDS, 1 % Triton X-100, 150 mM NaCl, 1 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0) and 20 mM Hepes (pH 7.6)) to a final concentration of 15x106 cells/ml. BrU-seq: U2OS osTIR1 or PAF1-AID cells were induced with doxycycline for 24 hrs, and subsequently with auxin for 5 hrs. After this treatment, cells were either mock-treated or irradiated with UV-C light (7 J/m2). Cells were then incubated in conditioned media for different periods of time (0, 3, 8, 24h) before being incubated with 2 mM bromouridine (Bru) at 37°C for a 30 min. The cells were then lysed in TRIzol reagent (Invitrogen) and Bru-containing RNA isolated as previously described (Andrade-Lima et al., 2015). ChIP-seq: Chromatin was sonicated to approximately 1 nucleosome using a Bioruptor waterbath sonicator (Diagenode). Chromatin of ~5x106 cells was incubated with 3ug antibody (RNAPII, rabbit polyclonal, Bethyl laboratories, A304-405A; PAF1, rabbit polyclonal, Bethyl laboratories, A300-172A) over night at 4°C, followed by a 1.5 hrs protein-chromatin pull-down with a 1:1 mix of protein A and protein G Dynabeads (Thermo Fisher; 10001D and 10003D). ChIP samples were washed extensively, followed by decrosslinking for 4 hrs at 65°C in the presence of proteinase K. DNA was purified using a Qiagen MinElute kit. Sample libraries were prepared using Hifi Kapa sample prep kit and A-T mediated ligation of Nextflex adapters. Samples were sequenced using an Illumina NextSeq500, using paired-end sequencing with 42 bp from each end. BrU-seq: cDNA libraries were made from the Bru-labeled RNA using the Illumina TruSeq library kit and paired-end 151 bp sequenced using the Illumina NovaSeq platform at the University of Michigan DNA Sequencing Core. Only single-end sequencing data was used for downstream analyses

Sequencing Platform

instrument_model
NextSeq 500

hg38

Number of total reads
26719666
Reads aligned (%)
96.0
Duplicates removed (%)
17.1
Number of peaks
2738 (qval < 1E-05)

hg19

Number of total reads
26719666
Reads aligned (%)
95.7
Duplicates removed (%)
17.3
Number of peaks
2705 (qval < 1E-05)

Base call quality data from DBCLS SRA